A. Nerve Growth Factor
A multi-component protein of molecular weight .about.130,000 has been isolated from mouse salivary glands, it being particularly concentrated in the glands of male mice, which is commonly referred to as "Nerve Growth Factor." The principal neural activity exhibited by the protein has been its ability to cause an increase in the size of sensory neurons, nerve cells which transmit impulses from sensory receptors to the brain, and in the size of sympathetic neurons, one of the two kinds of neurons which make up the autonomic nervous system which regulates the functional activity of the circulatory system, the glands, smooth muscles and other organs.
NGF as obtained by extraction at neutral pH from mouse salivary glands is known as 7S NGF and is made up of three subunits termed .alpha.-, .beta.-, and .gamma.-subunits. All of the neural activity of 7S NGF is exhibited by the .beta.-subunit, a dimer of two identical 118 amino acid peptides bound together by non covalent forces. This subunit is also referred to as 2.5S NGF. The .alpha.-subunit has no known biological activity. The .gamma.-subunit, however, is an arginine esteropeptidase. The initial genetic product in the synthesis of NGF is a prepro-NGF polypeptide which is cleaved by the .gamma.-subunit. The .gamma.-subunit has also been shown to accelerate wound healing in mice.
Recently, a third NGF component (M. wt. .about.116,000) has been reported to have been isolated from mouse salivary glands and to have shown to exhibit the property of being a plasminogen activator, i.e., it converts plasminogen to plasmin, suggesting its utility in the lysis of blood clots. See European Patent Application "Nerve Growth Factor and Process For Obtaining It" 78300656.2 (Publication No. 0002139Al) filed Nov. 22, 1978, published May 30, 1979.
As indicated above, the neural activity of NGF is exhibited by the .beta.-subunit (hereinafter .beta.NGF). It has been shown to stimulate markedly regenerative resprouting of transected axons of central adrenergic neurons, a property which makes it useful in the repair of damaged axons.
B. Recombinant DNA Technology
Recombinant DNA technology has reached the age of some sophistication. Molecular biologists are able to recombine various DNA sequences with some facility, creating new DNA entities capable of producing copious amounts of exogenous protein product in transformed microbes and cell cultures. The general means and methods are in hand for the in vitro ligation of various blunt ended or "sticky" ended fragments of DNA, producing potent expression vectors useful in transforming particular organisms, thus directing their efficient synthesis of desired exogenous product. However, on an individual product basis, the pathway remains tortuous and the science has not advanced to a stage where regular predictions of success can be made. Indeed, those who portend successful results without the underlying experimental basis, do so at considerable risk of inoperability.
DNA recombination of the essential elements, i.e., an origin of replication one or more phenotypic selection characteristics, an expression promoter, heterologous gene insert and remainder vector, generally is performed outside the host cell. The resulting recombinant replicable expression vector, or plasmid, is introduced into cells by transformation and large quantities of the recombinant vehicle are obtained by growing the transformant. Where the gene is properly inserted with reference to portions which govern the transcription and translation of the encoded DNA message, the resulting expression vector is useful to produce the polypeptide sequence for which the inserted gene codes, a process referred to as "expression." The resulting product may be obtained by lysis, if necessary, of the host cell and recovery of the product by appropriate purifications from other proteins.
In practice, the use of recombinant DNA technology can express entirely heterologous polypeptides--so-called direct expression--or alternatively may express a heterologous polypeptide fused to a portion of the amino acid sequence of a homologous polypeptide. In the latter cases, the intended bioactive product is sometimes rendered bioinactive within the fused, homologous/heterologous polypeptide until it is cleaved in an extracellular environment.
Similarly, the art of cell or tissue cultures for studying genetics and cell physiology is well established. Means and methods are in hand for maintaining permanent cell lines, prepared by successive serial transfers from isolated normal cells. For use in research, such cell lines are maintained on a solid support in liquid medium, or by growth in suspension containing support nutriments. Scale-up for large preparations seems to pose only mechanical problems.
Likewise, protein biochemistry is a useful, indeed necessary, adjunct in biotechnology. Cells producing the desired protein also produce hundreds of other proteins, endogenous products of the cell's metabolism. These contaminating proteins, as well as other compounds, if not removed from the desired protein, could prove toxic if administered to an animal or human in the course of therapeutic treatment with desired protein. Hence, the techniques of protein biochemistry come to bear, allowing the design of separation procedures suitable for the particular system under consideration and providing a homogeneous product safe for intended use. Protein biochemistry also proves the identity of the desired product, characterizing it and ensuring that the cells have produced it faithfully with no alterations or mutations. This branch of science is also involved in the design of bioassays, stability studies and other procedures necessary to apply before successful clinical studies and marketing can take place.